Revising mtDNA haplotypes of the ancient Hungarian conquerors with next generation sequencing

Abstract

As part of the effort to create a high resolution representative sequence database of the medieval Hungarian conquerors we have resequenced the entire mtDNA genome of 24 published ancient samples with Next Generation Sequencing, whose haplotypes had been previously determined with traditional PCR based methods. We show that PCR based methods are prone to erroneous haplotype or haplogroup determination due to ambiguous sequence reads, and many of the resequenced samples had been classified inaccurately. The SNaPshot method applied with published ancient DNA authenticity criteria is the most straightforward and cheapest PCR based approach for testing a large number of coding region SNP-s, which greatly facilitates correct haplogroup determination.

Fig 1. Chromatogram of two HVR-I sequence fragments of the Karos-III/16 sample from [9].

Arrows label double peaks, correct reads according to NGS data are listed above the arrows.

Fig 2. Electropherograms of two SNaPshot SBE-II reactions from two extracts of the same Karos-III/6 sample [9].

Characters at the top indicate Hg-s defined by the corresponding peaks. Black characters indicate peaks defining the ancestral allele, read characters indicate peaks defining the derived allele. Arrows point at double peaks. As each dye has a different influence on DNA mobility, positions of identical fragments with different dyes are not the same. Black arrows point at peaks taken into account, while blue arrows indicate neglected peaks, considered to have been derived from contamination. Orange peaks are size standards (GeneScan-120 LIZ, Applied Biosystems).